Rhein Inhibits AlkB Repair Enzymes and Sensitizes Cells to Methylated DNA Damage

Abstract

The AlkB repair enzymes, including Escherichia coli AlkB and two human homologues, ALKBH2 and ALKBH3, are iron(II)-and 2-oxoglutarate-dependent dioxygenases that efficiently repair N 1 -methyladenine and N 3 -methylcytosine methylated DNA damages. The development of small molecule inhibitors of these enzymes has seen less success. Here we have characterized a previously discovered natural product rhein and tested its ability to inhibit AlkB repair enzymes in vitro and to sensitize cells to methyl methane sulfonate that mainly produces N 1 -methyladenine and N 3 -methylcytosine lesions. Our investigation of the mechanism of rhein inhibition reveals that rhein binds to AlkB repair enzymes in vitro and promotes thermal stability in vivo. In addition, we have determined a new structural complex of rhein bound to AlkB, which shows that rhein binds to a different part of the active site in AlkB than it binds to in fat mass and obesity-associated protein (FTO). With the support of these observations, we put forth the hypothesis that AlkB repair enzymes would be effective pharmacological targets for cancer treatment.The nucleic acids in living cells are subject to modification by both endogenous and environmental agents (1). Direct-acting chemicals constantly damage nucleic acids and generate various methyl lesions with mutagenic and/or cytotoxic consequences (2, 3). O 6 -Methylguanine (O 6 mG) 2 and N 3 -methyladenine lesions have the highest potential for methylating damage by an SN1 agent such as N-methyl-NЈ-nitro-N-nitrosoguanidine (MNNG), which block replication and are thought to be toxic (4, 5). For the most part, the SN2 agent such as methyl methane sulfonate (MMS) produces N 1 -methyladenine (m 1 A) and N 3 -methylcytosine (m 3 C) lesions in single-stranded DNA (ssDNA). Accumulation of these adducts can lead to cell death (6, 7). Organisms have evolved several mechanisms to efficiently remove various methyl lesions, including suicidal methyltransferases, DNA glycosylases, and the AlkB family dioxygenases (see Fig. 1A) (8, 9). To date, AlkB repair appears to be the major natural defense mechanism with the power to restore the canonical base structure in vivo. Escherichia coli AlkB and its human homologues, ALKBH2 and ALKBH3, utilize iron(II) and 2-oxoglutarate (2OG) to achieve oxidative demethylation of m 1 A and m 3 C (see Fig. 1B) (10 -12). The lack of AlkB repair results in increased sensitivity to MMS, elevated level of mutations, and reduced cell proliferation (13-16). Furthermore, the accumulation of m 1 A and m 3 C lesions could also occur on RNA. Research suggests that the oxidative demethylation in mRNA and tRNA acts as a part of AlkB or ALKBH3 repair to protect cells against MMS (17, 18). The scope of substrates for AlkB repair has been largely extended to all simple N-alkyl lesions at the WatsonCrick base-pairing interface on the four bases (19), thus indicating the importance of oxidative demethylation for cell survival. In addition, human enzymes have been broadly linked to cancer. The housekeeping ...

The emerging roles of N6-methyladenosine (m6A) deregulation in liver carcinogenesis

“…Rhein, a natural product, is the first identified FTO inhibitor and competes with m6A-containing RNA for binding to the catalytic domain of FTO [97]. However, rhein is not an FTO-specific inhibitor, and it has been reported that rhein can also inhibit other ALKB family demethylases [98]. Meclofenamic acid (MA) is another FTO inhibitor and shows high selectivity in inhibiting FTO over ALKBH5 [99].…”

Structural Insights into N 6-Methyladenosine (m6A) Modification in the Transcriptome

“…It is of note that rhein and MA do not specifically inhibit FTO over other enzymes. For example, rhein also binds to 2OG-binding site of AlkB, a dioxygenase from Escherichia coli , and inhibits its activity [101] . As an anti-inflammatory drug, MA binds to the catalytic center of aldo–keto reductase 1C3 (AKR1C3) protein, which catalyzes the reduction of carbonyl groups on both steroids and prostaglandins in humans [102] .…”

Fluorescence Probes for ALKBH2 Allow the Measurement of DNA Alkylation Repair and Drug Resistance Responses

“…In a study of brain tumor samples taken from patients treated with alkylating drugs such as temozolomide (TMZ), all samples contained elevated levels of ALKBH2 mRNA over those from patients receiving radiotherapy . TMZ resistance in several glioblastoma cell lines as well as cisplatin resistance in H1299 lung cancer cells was suppressed by siRNA‐mediated knockdown of ALKBH2, and reduced ALKBH2 activity improves sensitivity to alkylating drugs . These findings have stimulated recent efforts to find inhibitors of ALKBH2 …”